Sliced Magnetic Polyacrylamide Hydrogel with Cell-Adhesive

3 Jun 2016 - In our previous work, we fabricated a novel magnetic hydrogel with ..... with Cell-Adhesive Microarray Interface: A Novel Multicellular S...
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Sliced Magnetic Polyacrylamide Hydrogel with Cell-Adhesive Microarray Interface: A Novel Multicellular Spheroid Culturing Platform Ke Hu,†,§ Naizhen Zhou,†,§ Yang Li,† Siyu Ma,† Zhaobin Guo,† Meng Cao,† Qiying Zhang,† Jianfei Sun,†,‡ Tianzhu Zhang,*,†,‡ and Ning Gu*,†,‡ †

State Key Laboratory of Bioelectronics, Jiangsu Key Laboratory for Biomaterials and Devices, School of Biological Sciences and Medical Engineering, Southeast University, Nanjing 210096, China ‡ Collaborative Innovation Center of Suzhou Nano-Science and Technology, Suzhou Key Laboratory of Biomaterials and Technologies, Suzhou 215123, China S Supporting Information *

ABSTRACT: Cell-adhesive properties are of great significance to materials serving as extracellular matrix mimics. Appropriate cell-adhesive property of material interface can balance the cell−matrix interaction and cell−cell interaction and can promote cells to form 3D structures. Herein, a novel magnetic polyacrylamide (PAM) hydrogel fabricated via combining magnetostatic field induced magnetic nanoparticles assembly and hydrogel gelation was applied as a multicellular spheroids culturing platform. When cultured on the cell-adhesive microarray interface of sliced magnetic hydrogel, normal and tumor cells from different cell lines could rapidly form multicellular spheroids spontaneously. Furthermore, cells which could only form loose cell aggregates in a classic 3D cell culture model (such as hanging drop system) were able to be promoted to form multicellular spheroids on this platform. In the light of its simplicity in fabricating as well as its effectiveness in promoting formation of multicellular spheroids which was considered as a prevailing tool in the study of the microenvironmental regulation of tumor cell physiology and therapeutic problems, this composite material holds promise in anticancer drugs or hyperthermia therapy evaluation in vitro in the future. KEYWORDS: magnetic hydrogel, cell-adhesive microarray interface, cell−matrix interaction, cell−cell interaction, multicellular spheroids, 3D cell culture

1. INTRODUCTION

vitro 3D model for both stem and tumor cell research, is usually generated by preventing cells adherent to matrix therefore favoring cell−cell interaction.11 Most matrices developed for generating multicellular spheroids were focused vitally on the anticell adhesive interfacial properties.12,13 The degree and distribution of cell adhesive sites on the interface of the matrix will greatly affect the cell behavior. Recent studies have indicated that a strong cell−matrix interaction between cells and 2D substrates induces cellular features that differ from those growing in vivo.14 It might be due to the competition between cell−cell and cell−matrix interaction which is greatly affected by the cell adhesive

Hitherto, monolayer cell culture model has provided many important results to interpret biological phenomena. However, research shows that cell behaviors are often unnatural when excised from native three-dimensional (3D) tissues and cultured on cell culture plates or Petri dishes.1,2 Findings in stem cell differentiation elucidate acute disparities in cell functions between 2D and 3D cell culture.3 Tumor cell cultures in 2D plates also display lower resistance to radiotherapy and chemotherapy compared with tumor cells in vivo.4 These deficiencies of traditional 2D culture models lead growing numbers of researchers to switch to develop novel materials for developing synthetic extracellular matrix (ECM) analogues.5−9 These matrices are designed on the basis of one or more structural or functional features of ECM to promote cells to form 3D structures.10 Multicellular spheroid, an important in © XXXX American Chemical Society

Received: April 8, 2016 Accepted: June 3, 2016

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DOI: 10.1021/acsami.6b04112 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 1. Schematic of sliced magnetic hydrogel applied as 3D cell culture matrix.

(0.2 μL) were mixed in 1 mL water first. The mixed solution was poured into a poly(tetrafluoroethylene) (PTFE) module after ultrasonic agitation for 10 min and then was subjected to a magnetostatic field. After a certain time for assembling, the gelation of polyacrylamide (PAM) was triggered by heating with a ceramic heating flake to 50 °C. The disorganized magnetic hydrogel was fabricated as the same procedure but in the absence of a magnetic field. Then, the obtained magnetic PAM hydrogel was fixed in an aluminum mold and was sliced with a low profile microtome blade. The final size of sliced magnetic hydrogel for 3D cell culture was 0.2 × 1.0 × 1.0 cm3. Before the biological experiments, the magnetic hydrogel was dialyzed for 2 days. 2.3. Morphology Characterization of Sliced Magnetic Hydrogel. Optical microscope images of sliced magnetic hydrogel and fluorescence microscope images were taken with a BX63 Olympus microscope. The optical microscope images were transferred into binary images with ImageJ, and the section areas of assemblies and the interval between magnetic nanoparticle assemblies were measured on the basis of these binary images with ImageJ. Atomic force microscopy (AFM) images were taken with a Bruker Dimension FastScan atomic force microscope. The sample was first replicated with polydimethylsiloxane (PDMS) and then was scanned with AFM. 2.4. Cell Culture. Sliced magnetic hydrogel first was rinsed with phosphate-buffered saline (PBS) twice and then was sterilized by immersion in ethanol solution (75%) for 24 h and in PBS for 24 h. Then, the sliced magnetic hydrogel was incubated with cell culture medium for 12 h before cell culturing. GFP-293A, MCF-7, and SK-OV-3 cells (1 × 106 cells/mL, 15 μL) in single cell suspension were grown on sliced magnetic hydrogel placed in 24-well cell-culture plates. Following initial plating of cells, they were allowed to adhere to the hydrogel before addition of complete growth medium to 3 mL. All cultures were maintained in an incubator at 37 °C in an atmosphere of 5% CO2. Live/dead cells were stained with fluorescein diacetate (FDA)/propidium iodide (PI) dye. Cell viability of tumor cells (SK-OV-3) cultured on cell culture plates and sliced magnetic hydrogel was characterized with cell counting kit-8 (CCK-8). 2.5. Time Lapse Microscopy. Time-lapse images of cells seeded on sliced magnetic hydrogel were acquired every 20 min at 100× magnification for 1 day using an X-living cell workstation (Olympus). 2.6. Laser Confocal Fluorescence Microscopy. Laser confocal fluorescence images were acquired at 100× magnification using SP8 (Leica) laser confocal fluorescence microscopy.

environment.15,16 We believe it is essential to balance cell−cell and cell−matrix interaction by controlling the interfacial adhesion properties for mimicking the ECM to build in vitro culture models. Researchers also found that multicellular hepatocyte spheroids could be formed when cultured in different natural materials by controlling the cell adhesive properties.17 Because of their ability to simulate the nature of most soft tissues, hydrogels capture numerous characteristics of the architecture and mechanics of the native cellular microenvironment. Recently, applying hydrogel-based composite materials as cell culture matrices drew the attention of many researchers. Among the diverse preparation strategies, the combination of nanomaterial and hydrogel holds promise of providing superior functionality.18,19 Interfacial properties of materials for cell culture have been implicated to play increasingly important roles on a wide spectrum of cellular functions.20 The heterogeneous interface of nanomaterial−hydrogel composite could affect cell behaviors and mimic ECM to a certain extent. In our previous work, we fabricated a novel magnetic hydrogel with anisotropic properties and controllable enhancement of magnetothermal effect when placing in the alternating magnetic field.21 In this study, we applied the slice of this multifunctional magnetic hydrogel as 3D cell culture matrix. Cell adhesive assemblies array combined with anticell adhesive substrate enhance the cell−cell interaction and promote the spontaneous formation of multicellular spheroid by both epithelial and cancer cells (Figure 1). This composite material could provide a new platform for different applications such as anticancer drugs or hyperthermia therapy models in vitro.

2. MATERIALS AND METHODS 2.1. Materials. Polyglucose sorbitol carboxymethyether encapsulated Fe3O4 magnetic nanoparticles (Fe3O4@PSC MNPs) were provided by Jiangsu Key Laboratory for Biomaterials and Devices. Millipore quality water (18.25 MΩ cm−1), prepared with a Milli-Q Plus water system, was the only used water throughout experiments. Human embryonic kidney 293A (stably transfected with enhanced green fluorescent protein encoding gene (EGFP)) was established and provided by Jiangsu Key Laboratory for Biomaterials and Devices. Breast cancer cell line MCF-7 and ovarian cancer cell line SK-OV-3 were purchased from Chinese Academy of Science Shanghai cell bank. Unless stated, all reagents were purchased from Aladdin Industrial Inc. 2.2. Preparation of Sliced Magnetic Hydrogel. The preparation of anisotropic magnetic hydrogel has been described previously.18 Briefly, Fe3O4@PSC MNPs, acrylamide monomer (87 mg), N,N′-methylene-bis-acrylamide (9 mg), ammonium persulfate (2.4 mg), and tetraethylethylenediamine B

DOI: 10.1021/acsami.6b04112 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces 2.7. Drug Cytotoxicity Analysis. To establish dose response of cells on monolayer and sliced magnetic hydrogel, cells were treated with 2, 25, and 100 μg/mL doxorubicin in standard medium after a given culture period. Cytotoxicity was evaluated following a 24 h incubation on both monolayer and sliced magnetic hydrogel.

percentage of assemblies in the section area of the sliced magnetic hydrogel was calculated. The results demonstrated that structure properties of the assemblies’ array (assemblies’ number and sectional areas) on the surface were of no significant difference between each sample with different slicing depths and showed a good reproducibility (Supporting Information, Figure s3). As in Figure 3, the area of one assembly, which largely distributed around 4−12 μm2, grew slightly when the concentration of nanoparticles increased. According to Figure 3a, the peak value emerges at areas of 4, 8, and 12 μm2 in SMH-1, SMH-2, and SMH-3, respectively. At lower nanoparticle concentrations (SMH-1 and SMH-2), the ratio of assemblies in the area ranging from 4 to 8 μm2 is near 90%, while at higher concentrations, more than 60% of assemblies are in the range of 8−12 μm2. Furthermore, as shown in Figure 3b, the concentration impacts the interval between assemblies significantly. The distribution of the interval of SMH-1 is quite wide, from 10 to 40 μm. With the concentration increasing, the distribution becomes narrower and the interval between assemblies of SMH-3 only ranges from 5 to 10 μm, which showed that with the increasing of concentration the degree of homogeneity of assemblies also improved. 3.2. Sliced Magnetic Hydrogels Applied as 3D Cell Culture Matrix. Previous studies showed that cell−cell interaction played an important role in cell behaviors and functions.23 Strong cell−matrix interaction inhibited the formation of cell−cell interaction when cells were cultured on 2D plates. On the basis of this, researchers fabricated hydrogels and electrospinning scaffolds with limited cell-adhesive sites to promote the cell−cell interaction, and several cancer cell lines were able to form multicellular spheroids when cultured on these matrices.24−26 Sliced magnetic hydrogels composed of cell-adhesive nanoparticle assemblies and anti-cell-adhesive hydrogels also capable of providing a matrix with cell-adhesive properties more possibly accord with the ECM than the traditional monolayer culture plate. Since cells cannot adhere on the hydrophilic surface of the PAM hydrogel, the celladhesive function of magnetic hydrogels is provided by the magnetic nanomaterials. However, we found that very few cells could adhere on the surface of disorganized sliced magnetic hydrogels that were fabricated in the absence of a magnetic field (Supporting Information, Figure s4). This might be because the array consisted of randomly distributed magnetic nanoparticles that were too small to support cells to form focal adhesion.27 Compared to the unassembled magnetic nanoparticles, the sectional areas of magnetic colloidal assemblies were large enough for a single cell to form focal adhesion. Moreover, magnetic nanoparticles of the colloidal assemblies were less likely to be encapsulated inside the hydrogel after slicing. Since the magnetic hydrogels were sliced before being applied as cell culture matrix, the roughness of the sliced surface was characterized by atomic force microscope in the area of 400 μm2 (20 μm × 20 μm), which is close to the area of one single cell after molding with polydimethylsiloxane (PDMS) (Supporting Information, Figure s5). Generally, the heights of the peaks and valleys ranged from tens to hundreds of nanometers, while specifically the surface roughness (Ra) was 422 ± 169 nm, which is quite close to a polished surface.28 Research showed that surface roughness ranging from one micrometer to tens of micrometers would affect the behavior of cells.29 The roughness of magnetic hydrogels caused by slicing was out of this range (both larger and smaller than) and had

3. RESULTS AND DISCUSSION 3.1. Fabrication and Characterization of Sliced Magnetic Hydrogel. In our experiments, owing to good biocompatibility and magnetism, Fe3O4 nanoparticles coated by polyglucose sorbitol carboxymethyether (Fe3O4@PSC) were used as the building block (Supporting Information, Figure s1).22 The average hydrodynamic diameter of Fe3O4@PSC nanoparticles was 201 nm, and the magnetic force on this nanomaterial can effectively overwhelm the thermal perturbation on the basis of our calculation.18 PAM hydrogel was chosen as the host material because it is biocompatible after dialysis and because the mechanical property of hydrogel is suitable as a mimic for ECM.14 By combining static magnetic field-assisted assembly of magnetic nanoparticles and gelation together, we fabricated this magnetic hydrogel with anisotropic properties. Since the assemblies were encapsulated inside the hydrogel, magnetic hydrogels were sliced in order to expose assemblies on the interface. The sample (1.0 cm × 1.0 cm × 1.0 cm) was fixed in a mold to avoid break caused by deformation of anisotropic hydrogel when slicing, and it was sliced with a low profile microtome blade. The final size of sliced magnetic hydrogel for 3D cell culture was 0.2 cm × 1.0 cm × 1.0 cm. A series of sliced magnetic hydrogels with different concentrations of Fe3O4 nanoparticles (0.05, 0.3, and 1.8 mg/ mL) were fabricated and labeled as SMH-1, SMH-2, and SMH3, respectively. The increase of concentration of Fe3O4 nanoparticles could be easily distinguished with naked eyes (Figure 2a), and the optical microscope images of the side view

Figure 2. (a) Photographs and (b) optical microscope images of sliced magnetic hydrogel samples SMH-1, SMH-2, and SMH-3 (from the left to the right). Scale bar in b: 10 μm.

of sliced magnetic hydrogels accorded with this tendency (Supporting Information, Figure s2). The section morphologies of sliced magnetic hydrogels with Fe3O4 nanoparticles as building blocks are characterized with an optical microscope (Figure 2b). Previous studies showed that the assembly process could promote the homogeneity of the distribution of nanomaterials inside the hydrogel.23 The optical microscope images of sliced magnetic hydrogels were analyzed with image processing software. Specifically, sectional areas of assemblies and intervals between assemblies were measured, and the C

DOI: 10.1021/acsami.6b04112 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 3. (a) Statistical diagrams of assembly sectional area and (b) intervals between magnetic nanoparticle assemblies of sliced magnetic hydrogel samples SMH-1, SMH-2, and SMH-3 (from the left to the right).

the concentration of magnetic nanoparticles increased. On the basis of Figure 3a, the cell-adhesive area of a sliced magnetic hydrogel increased from approximately 0.9% to 17% when the concentration of magnetic nanoparticles rose from 0.05 mg/mL to 1.8 mg/mL and when the density of the adhesive site was still not large enough to prevent cells from forming multicellular spheroids.17 However, further increasing the density of magnetic nanoparticles will induce the aggregate and precipitate of nanoparticles when mixed with the polymer monomer solution. This makes it not possible to fabricate magnetic hydrogels containing a higher concentration of magnetic nanoparticles. Considering the suitable concentration of magnetic nanoparticles required for alternating magnetic field induced hyperthermia of magnetic hydrogels,32 we chose SMH-2 for further evaluation. Results showed that GFP-293A cells overlaid as single-cell suspension on SMH-2 formed 3D cell multicellular spheroids 1 day after plating (Figure 4a), and the average diameter and total numbers of multicellular spheroids clearly increased 6 days postplating (Figure 4b), which was not observed with the GFP-293A cells cultured in 24-well plates (Figure 4c). Three-dimensional reconstruction images obtained with a laser confocal fluorescence microscope showed that multicellular spheroids were not formed inside the sliced

little impact on cells. Consequently, we believe that the sliced surface of magnetic hydrogels could be regarded as a smooth surface to cells.30 To evaluate the performance of sliced magnetic hydrogel as 3D cell culture platform, human embryonic kidney 293A (stably transfected with enhanced green fluorescent protein encoding gene (EGFP)) was chosen because 293A cells tended to form a multicellular spheroid in traditional nonadhesive 3D culture model. A GFP-293A single cell suspension (15 μL of 1 × 106 cells/mL) was pipetted onto the surface of the composite material placed in a 24-well plate. After 1 h incubation in CO2 incubator, 3 mL culture medium was added. According to the optical microscope images, the proportion of cell-adhesive site areas on the surface of sliced magnetic hydrogels rose with increasing concentration of magnetic nanoparticles. Sliced magnetic hydrogels with a concentration of Fe3O4 nanoparticles higher than 0.3 mg/mL were light-tight, and the morphology of cells cultured on these composite materials could hardly be observed with an inverted microscope. Thus, the cell cultured on sliced magnetic hydrogels with concentrations of Fe3O4 nanoparticles higher than 0.4 mg/ mL was observed after the composite material was overturned. After preliminary experiments, we found that GFP-293A cells cultured on all three samples with different densities of celladhesion sites could form multicellular spheroids after 6 days (Supporting Information, Figure s6). The number and size of the multicellular spheroids formed on SMH-1, SMH-2, and SMH-3 were counted and measured (Supporting Information, Figure s7). Research showed that the size and size distribution of multicellular spheroids were cell-line dependent and that the numbers of multicellular spheroids formed increased with the density of adhering cells caused by partially exposed Fe3O4 nanoparticles.12,31 Results showed that the size of multicellular spheroids formed on different samples were of no significant difference. The numbers of multicellular spheroids formed on sliced magnetic hydrogels per sample slightly increased when

Figure 4. Florescent microscope image of GFA-293A cells cultured on sliced magnetic hydrogel sample SMH-2 for (a) 1 day and (b) 6 days and (c) on 24-well plate for 6 days. Scale bar: 100 μm. D

DOI: 10.1021/acsami.6b04112 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

different cell lines were obviously different. Researchers found that many tumor cell lines could only form loose aggregates, an architecture that loses tight cell−cell conjunction, thereby significantly different from the in vivo tumor features.34 We chose breast cancer cell line MCF-7 and ovarian cancer cell line SK-OV-3 as models to study the multicellular spheroid formation capability on the sliced magnetic hydrogel. Results showed that after 3 days culture, MCF-7 cells initially formed a large amount of small cell aggregates (Figure 6a). These cell

magnetic hydrogel but on the surface (Supporting Information). These multicellular spheroids will not drop off when undergoing a gentle shaking. To further investigate the formation process of multicellular spheroids on the surface of sliced magnetic hydrogels, an Olympus X-living cell workstation was used to acquire the time-lapse images of seeded cells (cellseeding density was doubled to accelerate the spheroid formation) every 20 min at 100× magnification for 1 day (Supporting Information). Results demonstrated that GFP293A cells initially formed small aggregates via proliferation and migration, and then these aggregates further merged into irregular spheroids and, finally, became mature with inerratic geometry (Figure 5). The colocalization of magnetic nano-

Figure 6. Optical microscope images of MCF-7 cells and SK-OV-3 cells cultured on sliced magnetic hydrogels for (a, e) 3, (b, f) 6, (c) 9, and (g) 12 days. Live/dead cells were stained with PDA (green)/PI (red) (d, h). Scale bar: 50 μm.

aggregates continued to grow and merge to form multicellular spheroids after 6 days culture (Figure 6b). Results of live/dead staining with fluorescein diacetate (FDA) and propidium iodide (PI) demonstrated that most dead cells were located in the center of partial multicellular spheroids after 9 days culture (Figure 6c, d), which should be induced by the diffusion limitation of nutrition and metabolic wastes. SK-OV-3 cells cultured on sliced magnetic hydrogels presented similar results (Figure 5e, f), although dead cells (red) were observed after being cultured for 12 days (Figure 6g, h). The proliferation of tumor cells in monolayer was comparably faster than those cultured in 3D.10 Results showed that the proliferation rate of SK-OV-3 cultured on sliced magnetic hydrogels was clearly higher than those cultured in monolayer (Supporting Information, Figure s11), which was close to the growth rate in vivo.35 However, when cultured in hanging drop system, SKOV-3 cells could only form cell aggregates with no penetration resistance (Supporting Information, Figure s12). The drug resistance of SK-OV-3 cells cultured on the sliced magnetic hydrogel and in the culture plates was preliminarily evaluated with doxorubicin as a model drug. Results demonstrated that the cells cultured on sliced magnetic hydrogels presented stronger drug resistance than those cultured on plates when the concentration of doxorubicin increased to 100 μg/mL after culturing for 9 days, which was due to the compact structures of multicellular spheroids (Supporting Information, Figure s13). Previous studies showed that SK-OV-3 cells could form multicellular spheroids and showed drug resistance when cultured inside RGD-modified, cell-secreted metalloproteinases (MMP) sensitive hydrogel.10 Furthermore, research demonstrated that adding extracellular adhesion molecules (fibronectin, laminin, or reconstituted basement membrane) into the hanging drop culture system could induce SK-OV-3 cells to form multicellular spheroids.36 Combined with our findings, we speculate that the matrix with appropriate cell-adhesion properties is necessary for the formation of multicellular spheroid.37 Further studies on the cytobiology mechanism of

Figure 5. (a−f) Time-lapse microscope images of GFP-293A cells cultured on sliced magnetic hydrogel. Blue arrows point to the spontaneous formation process of multicellular spheroids via migration, proliferation, and mergence. Scale bar: 100 μm.

particle assemblies and multicellular spheroids was demonstrated with optical microscopy images at low exposure. Results showed that GFP-293A cells started to aggregate 2 h after seeding (Supporting Information, Figure s8). Intervals among assemblies were smaller than the size of the single cell and were able to be stepped over easily by cells. After 2 days, multicellular spheroids had formed and localized on top of the nanoparticle assemblies. Since the diameter of multicellular spheroids ranged in hundreds of micrometers, the optical microscope could not focus on the surface of the sliced magnetic hydrogel and multicellular spheroids at the same time (Supporting Information, Figure s9). After culturing for 6 days, with the growing of those spheroids, they became opaque; therefore, the nanoparticle assemblies underneath became invisible (Supporting Information, Figure s10). However, the assemblies around the multicellular spheroids proved that those areas underneath the spheroid certainly had assemblies exposed, owing to the homodistribution of assemblies that area with no assemblies surrounded by area with assemblies does not possibly exist. The spontaneous formation processes of multicellular spheroids, cell morphology, and behaviors were similar to cell culture on staple commercial materials (such as Matrigel),33 but costs of sliced magnetic hydrogels were much lower and had almost no batch-to-batch discrepancy. Furthermore, the spheroid formation efficiency of cells cultured on sliced magnetic hydrogels was also no worse than in developed techniques.34 Most fully fledged techniques used to generate multicellular spheroids (such as hanging drops) nowadays were designed on the basis of the nonadhesion strategy. Findings in these models demonstrated that the spheroid formation capabilities among E

DOI: 10.1021/acsami.6b04112 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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support from the research innovation plan of colleges and universities postgraduates of Jiangsu Province (CXLX12_0120).

spheroid formation could be carried out by applying this composite material as platform.

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4. CONCLUSION In summary, a novel sliced magnetic hydrogel with certain regularity and reproducibility was prepared and applied as 3D cell culture platform. By combining anti-cell-adhesive hydrogel and cell-adhesive magnetic colloidal assemblies together, this composite material provided a matrix with cell-adhesive microarray on the surface which could enhance the cell−cell interaction. Cells from different cell lines could spontaneously form multicellular spheroids when cultured on this functionalized interface, and on this platform, cells which could only form cell aggregates in traditional 3D cell culture system were able to form multicellular spheroids, which promote the applicability of this matrix. This low-cost magnetic hydrogel will have a great potential in in-vitro evaluation of hyperthermia and chemotherapy or cytobiology research.

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(1) Cukierman, E.; Pankov, R.; Stevens, D. R.; Yamada, K. M. Taking Cell-Matrix Adhesions to the Third Dimension. Science 2001, 294 (5547), 1708−1713. (2) Even-Ram, S.; Yamada, K. M. Cell Migration in 3D Matrix. Curr. Opin. Cell Biol. 2005, 17, 524−532. (3) Tibbitt, M. W.; Anseth, K. S. Hydrogels as Extracellular Matrix Mimics for 3D Cell Culture. Biotechnol. Bioeng. 2009, 103, 655−663. (4) Fitzgerald, K. A.; Malhotra, M.; Curtin, C. M.; O’ Brien, F. J.; O’ Driscoll, C. M. Life in 3D is Never Flat: 3D Models to Optimize Drug Delivery. J. Controlled Release 2015, 215, 39−54. (5) Nyga, A.; Cheema, U.; Loizidou, M. 3D Tumour Models: Novel In Vitro Approaches to Cancer Studies. J. Cell Commun. Signal. 2011, 5, 239−5248. (6) Kraehenbuehl, T. P.; Langer, R.; Ferreira, L. S. ThreeDimensional Biomaterials for the Study of Human Pluripotent Stem Cells. Nat. Methods 2011, 8, 731−736. (7) Zhang, P.; Wang, S. Designing Fractal Nanostructured Biointerfaces for Biomedical Applications. ChemPhysChem 2014, 15, 1550−1561. (8) Sun, K.; Liu, H.; Wang, S.; Jiang, L. Cytophilic/Cytophobic Design of Nanomaterials at Biointerfaces. Small 2013, 9, 1444−1448. (9) Liu, X.; Wang, S. Three-dimensional Nano-biointerface as a New Platform for Guiding Cell Fate. Chem. Soc. Rev. 2014, 43, 2385−2401. (10) Loessner, D.; Stok, K. S.; Lutolf, M. P.; Hutmacher, D. W.; Clements, J. A.; Rizzi, S. C. Bioengineered 3D Platform to Explore Cell−ECM Interactions and Drug Resistance of Epithelial Ovarian Cancer Cells. Biomaterials 2010, 31, 8494−8506. (11) Ziqi, Z.; Jianjun, G.; Yening, Z.; Ying, G.; Zhu, X. X.; Yongjun, Z. Hydrogel Thin Film with Swelling-induced Wrinkling Patterns for High-throughput Generation of Multicellular Spheroids. Biomacromolecules 2014, 15, 3306−3312. (12) Yukie, Y.; Atsuo, W.; Kaori, Y.; Anna, K.; Maki, K.; Hideo, N.; Yusei, K.; Yasushi, K.; Hiroshi, Y.; Takako, F.; Tatsuya, A.; Hidehiko, O.; Juri, G. G.; Yasuhisa, F. The Use of Nanoimprinted Scaffolds as 3d Culture Models to Facilitate Spontaneous Tumor Cell Migration and Well-regulated Spheroid Formation. Biomaterials 2011, 32, 6052− 6058. (13) Rizvi, I.; Celli, J. P.; Evans, C. L.; Abu-Yousif, A. O.; Muzikansky, A.; Pogue, B. W.; Finkelstein, D.; Hasan, T. Synergistic Enhancement of Carboplatin Efficacy with Photodynamic Therapy in a ThreeDimensional Model for Micrometastatic Ovarian Cancer. Cancer Res. 2010, 70 (22), 9319−9328. (14) Engler, A.; Bacakova, L.; Newman, C.; Hategen, A.; Griffin, M.; Discher, D. Substrate Compliance versus Ligand Density in Cell on Gel Responses. Biophys. J. 2004, 86 (1), 617−628. (15) Douezan, S.; Dumond, J.; Brochard-Wyart, F. Wetting transitions of cellular aggregates induced by substrate rigidity. Soft Matter 2012, 8 (17), 4578−483. (16) Ryan, P. L.; Foty, R. A.; Kohn, J.; Steinberg, M. S. Tissue Spreading on Implantable Substrates is a Competitive Outcome of Cell−Cell vs. Cell−Substratum Adhesivity. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 4323−4327. (17) Powers, M. J.; Rodriguez, R. E.; Griffith, L. G. Cell-substratum Adhesion Strength as a Determinant of Hepatocyte Aggregate Morphology. Biotechnol. Bioeng. 1997, 53 (4), 415−426. (18) Dvir, T.; Timko, B. P.; Brigham, M. D.; Naik, S. R.; Karajanagi, S. S.; Levy, O.; Jin, H.; Parker, K. K.; Langer, R.; Kohane, D. S. Nanowired Three-Dimensional Cardiac Patches. Nat. Nanotechnol. 2011, 6, 720−725. (19) Ding, X.; Liu, H.; Fan, Y. Graphene-Based Materials in Regenerative Medicine. Adv. Healthcare Mater. 2015, 4, 1451−1468. (20) Ayala, R.; Zhang, C.; Yang, D.; Hwang, Y.; Aung, A.; Shroff, S. S.; Arce, F. T.; Lal, R.; Arya, G.; Varghese, S. Engineering the Cell-

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b04112. The morphology and magnetic properties of Fe3O4@ PSC MNPs; the optical microscope images of the side view of a sliced magnetic hydrogel and the sectional view of a sliced magnetic hydrogel with different slicing depths; AFM characterization of a sliced magnetic hydrogel; statistical diagram of numbers and size of multicellular spheroids formed on SMH-1, SMH-2, and SMH-3; morphology of GFP-293A cells cultured on sliced disorganized magnetic hydrogels, SMH-1 and SMH-3; morphology of SK-OV-3 cells cultured in hanging drop system (PDF) 3D reconstruction images obtained with laser confocal fluorescence microscope (AVI) Time-lapsed video of GFP-293A cells cultured on SMH2 (AVI)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Fax: +86 25 83272460. *E-mail: [email protected]. Fax: +86 25 83272460. Author Contributions §

These two authors contributed equally in this manuscript. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the support of National Natural Science Foundation of China (61127002, 61420106012). T. Z. Zhang is grateful for support from the program for New Century Excellent Talents in University (NCET-09-0298) and Suzhou medical apparatus and new medicine Fund (ZXY201440), and J. F. Sun is thankful for support from National Natural Science Foundation of China ( 21273002). K. Hu is also grateful for F

DOI: 10.1021/acsami.6b04112 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

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DOI: 10.1021/acsami.6b04112 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX